摘要
目的：获得新的鼠抗人黑色素瘤单抗ＨＢ８７５９轻、重链可变区基因，以便对其进行基因工程改造．方法：采用反转录－ＰＣＲ法（ＲＴ－ＰＣＲ）从杂交瘤细胞中扩增抗体轻、重链可变区基因，测定ＤＮＡ序列，输入计算机分析，并在大肠杆菌中表达．结果：在ＥＭＢＬＧｅｎｅＢａｎｋ１９９５中未发现有与轻、重链可变区（ＶＨ，ＶＬ）基因序列相同的基因．ＶＨ，ＶＬ基因均为单一开放读框，并具有抗体可变区结构特征，ＶＨ，ＶＬ基因分别长３６６ｂｐ，３２４ｂｐ．将ＶＨ，ＶＬ基因重组入融合蛋白表达载体ｐＧＥＸ－４Ｔ－１，诱导后可见ＶＨ，ＶＬ分别在４０ｋｕ和３８ｋｕ处有深染的新蛋白带出现，与预计的ＶＨ，ＶＬ与ＧＳＴ的融合蛋白的分子质量相符．结论：ＶＨ，ＶＬ基因均为新发现的基因序列，符合功能重排的鼠抗体可变区基因特征．
Objective: To acquire new variable region genes of anti human melanoma murine monoclonal antibody HB8759. Methods: Reverse transcriptional PCR was applied to amplify heavy and light chain variable region genes of the mAb from hybridoma cells. The two genes were sequenced, analyzed by computer and expressed in E.coli . Results: No genes identified as the same as the V H and V L genes were found in EMBL Gene Bank 1995. V H gene and V L gene consisted of 366 and 324 base pairs respectively. Both V H and V L genes were open reading frames and had antibody characteristics. The two genes were inserted into expression vector pGEX 4T 1. The results showed that GST V H and V L fusion proteins were of anticipated sizes. Conclusion: V H and V L genes are confirmed as functionally rearranged mouse immunoglobulin variable region genes and appeared to be new genes.
出处
《第四军医大学学报》
1997年第5期404-408,共5页
Journal of the Fourth Military Medical University
